基于位点特异性PCR技术对眼镜王蛇的分子鉴定研究

Identification of Ophiophagus hannah by Allele-Specific PCR with Molecular Methods

关键词:  眼镜王蛇  特异性聚合酶链式反应  细胞色素C氧化酶亚基Ⅰ序列  特异性单核苷酸多态性变异位点  特异性引物

Key words:  Ophiophagus  hannah, specific  polymerase chain  reaction,Cytochrome  OxidaseⅠ sequences,specific  single nucleotide  polymorphisms,specific  primers

摘要:

目的 建立一种眼镜王蛇特异性聚合酶链式反应鉴别方法。方法 收集眼镜王蛇、眼镜蛇、蕲蛇等21种蛇类,通过比对21种蛇类的细胞色素C氧化酶亚基Ⅰ序列,寻找眼镜王蛇特异性单核苷酸多态性变异位点,设计特异性引物；通过对循环次数、退火温度等关键因素的考察,确立最优反应体系,并将方法应用到眼镜王蛇粉末中进行药材粉末的基原鉴别。结果 当退火温度为58 ℃,循环次数为30次时,眼镜王蛇在约200 bp处有特异性条带,而其他蛇类无此条带。结论 该方法能够将眼镜王蛇与其他蛇类区别,且能鉴别眼镜王蛇粉末的基原,为眼镜王蛇药材及其饮片质量评价体系的制定和完善提供科学依据。

Abstract:

OBJECTIVE To establish a specific polymerase chain reaction idetification of Ophiophagus hannah. METHODS A total of 21 samples were collected,including Ophiophagus hannah,Naja naja,Agkistrodon acutus,etc. By comparing the Cytochrome OxidaseⅠsequences of 21 snake samples,the specific single nucleotide polymorphisms of Ophiophagus hannah were searched and specific primers were designed;the key factors such as the number of cycles,annealing temperature were investigated to established the optimal reaction system, The method was applied to the medicinal primordial identification of Ophiophagus hannah. RESULTS When the annealing temperature was 58 ℃ and cycles number was 30,a specific band of 200 bp was amplified in the Ophiophagus hannah with Specific primers, while other snake samples has no specific band. CONCLUSIONS This method can distinguish the Ophiophagus hannah from other snake samples,and identify the primordial of the Ophiophagus hannah powder. It provides a scientific basis for the formulation and improvement of the quality evaluation system of Ophiophagus hannah medicinal materials and its decoction piece.